Welcome !!!FISH 543 / OCEAN 575

Molecular Techniques

Introductions

• Danielle Mitchell– Room MAR 175

– mitcheld@u.washington.edu

– Office hours: By appointment

• Dr Lorenz Hauser– Room MAR 207

– Tel: 685-3270

– lhauser@u.washington.edu

– Office hours: By appointment

MMBL Marine Molecular Biotechnology Lab

• 4 faculty– 2 fisheries (Naish, Hauser)

– 2 oceanography (Armbrust, Rocap)

• Main research areas– Conservation Genetics

– Molecular Ecology

– Genomics

– Phytoplankton Ecology

• Come and talk to people!We are

hereMMBL

Introduce yourself

• Name• Home Department• Project

– Background

– Specific question

– Molecular methods

Pairs

• Similar projects– Can share some tasks

• buffers, extraction, cloning etc.

• Lab today

• Suggested pairs– Jim Franks – Pilar Montepan

– Brian Kristall – Gang Xin

– Kristi Straus – Nick Adams

– Thomas Unfried – Bill Webb

– Danny Garrett – Alfred Sidman

Some General Info• Prerequisite

– FISH 542 / OCEAN 574• Check web page

– Username – Ocean574– Password - Gryffindor

• Will present some information • Read!

– Hillis et al. (1996) Molecular Systematics. Sinauer– References on 542 homepage

• Ask

• Textbook– Guidebook ‘Maniatis’

• Sambrook & Russell et al. 2001• Copy in lab – LEAVE IT THERE!!

Communication

• Check the homepage frequently– Subject to change

– Uploaded:• Lecture slides

• Protocols of general interest

• Links

• Papers

– Contribute• Links

• Protocols

• Lab meeting– Tuesdays 1:30

• Ask questions– In labs

– Arrange meetings

• E-mail– To whole class

– To us

• Talk to each other– Class mates

– Students in MMBL

Course description

• Initial training– Set lab exercises– Designed to show general

procedures• Not only own project

• Lab access– Two sessions

• Tuesdays & Thursdays

– Open access other times• Recommended to finish

projects• During building opening

hours• Will get keycode lock

• Lab Meetings• Facilities

– Main lab• FTR 129

– Meetings• FTR 103

– Some equipment• MMBL tour

Facilities• FTR 129

– Main lab– Bench space, extraction,

electrophoresis etc.

• FTR 103– Meetings– Food and drink

• MMBL– Marine Studies Building– Some equipment

• Sequencer• Gel scanner

– Emergency supplies• Let us know!

• Computers– Three in the lab– More PCs in FSH 207 &

FSH 209– Macs

• ask

– Software links on webpage

Safety

• No eating or drinking in lab– Use FTR 103

• Wear labcoats and gloves– Goggles and masks for some materials– No open shoes

• Assume all materials are hazardous– Many are– If in doubt, consult MSDS

• links on webpage

– Particularly nasty stuff will be flagged• Some on webpage

• Spillages– Report to instructors

• Electrophoresis– Switch off before touching

• If in doubt -ask us

Assessment

• Detailed description on homepage

• Proposal (10%)– 5 pages

– Example on web

– Due end of next week• Friday Jan 16, 2004

• Proposal review– Two colleagues

• Be nice but critical

– Instructor

– Due end of week 3• Friday Jan 23, 2004

Item %

Research Proposal

10

Proposal Review 10

Lab Notebooks 20

Lab Participation 15

Discussion Participation

15

Presentation 10

Report 20

Assessment

• Lab Notebook– Essential part of the course

• Reproducibility

• Can stick in protocols

• Provide details and describe deviations

– Use bound lab notebooks• Ring binders lose leaves

– Will collect lab notebooks 2-3 times during the quarter

• Lab Participation– Show up!

• Tuesdays & Thursdays

– Try hard!

• Discussion participation– Will meet every Tuesday

Item %

Research Proposal

10

Proposal Review 10

Lab Notebooks 20

Lab Participation 15

Discussion Participation

15

Presentation 10

Report 20

Assessment

• Presentation– Meeting in final week

• Tuesday March 17– Invite MMBLers

• Present results– 10 min– Future work

• Report– 8 pages

• Incorporate comments on talk and proposal

– Scientific paper format• Introduction• Methods• Results• Discussion

– Due Thursday March 19

Item %

Research Proposal

10

Proposal Review 10

Lab Notebooks 20

Lab Participation 15

Discussion Participation

15

Presentation 10

Report 20

The proposal

• Needed because FISH 542 / OCEAN 574 not offered

• Main Aims– Sort out ideas

• Based on background

– Define questions

– Check feasibility

– Timeline

– Wider significance

• Dissertation projects– Can be used (not verbatim if it’s

not yours)

– Concentrate on work in class

– Will be considered in assessment

• Budget– Idea of how much it is

– Estimate of additional funds required

Sample collection and storage; DNA extraction and storage

Tissue collection

• Almost any living tissue

• Preferably fresh organisms– Less stringent (ancient DNA)

• Quantity less important – Can get DNA out of single cells

– more important is ratio tissue / preservative

• Non-destructive sampling– feathers, hairs, feces

– Contain cells or cell debris

Tissue preservation

• DNA– inhibit enzyme activity of enzymes eating DNA

– Usually accomplished by dehydration or freezing• alcohol, high salt concentration or drying

• Alcohol is most commonly used technique

• Small material (cells): frozen in TE buffer

– Formalin preservation is no good• Preferred method in museums

• causes degradation and irreversible cross-linking.

• Some protocols are at http://www.public.iastate.edu/~curteck/Formalin_Fixed_DNA.htm

Tissue storage• Important issue

– Reproducing results– Temporal changes– Endangered species

• Three main ways– Freezing

• Burke museum genetic specimen collection• Problem: equipment or power failure

– Ethanol• Keep below room temperature• Explosive !

– Drying• Herbarium specimen

• DNA is surprisingly stable– Extracted from very old specimen– But – only some markers can be applied

DNA extraction – the basic concept

Cell lysis

Protein removal

DNA precipitation

Process Common procedure

•SDS, sarcosyl•CTAB

•Proteinase K•Lysozyme

•Freezing•Sonication•Grinding

Chemical

Enzymatic

Mechanic

Alcohol •Ethanol•Iso-propanol

•Phenol•chloroform

•Sodium chloride•Sodium acetate

•Membrane•Beads

Organic solvents

Salt

DNA binding

Many variations on the theme• Depending on DNA quality and throughput required

– Problem often degradation • DNA in small fragments

– Most PCR based methods get away with bad DNA• Hotshot (Biotechniques 29:52 (2000))

– Boil it and PCR it

• Salting out

• But often PCR inhibitors– Co-purify with DNA

• humic acids• Secondary cell components in plants

– Can be tested by adding extract to working PCR

• Ways to further purify DNA– Ultracentrifugation in CsCl gradients– Gel electrophoresis– Chromatography

• Rapid development of methods– Talk to people– Newsgroups– Company newsletters

Commercial kits

• Bind DNA selectively to a membrane

• Wash rest away

• Many specific commercial kits– Plant– Animals– Soil– Feces

DNA storage• Difficult but important field

– DNA techniques only in 30 years• Little experience with long term storage

– Development of new markers• Verify results

• Compare markers

– Temporal studies• Endangered species

• Climate change

• Anthropogenetic change

• DNA is very stable– May survive up to 100,000 years

– Drosophila paper (Colton & Clark 2001)• Six different storage methods (2 years)

• Three extraction methods

• PCR of 800 bp fragment it ain’t matter

• Lots of reports of degraded, unusable DNA

DNA storage• Main options

– Salt solution• EDTA – prevents enzyme activity

– Only short term

• Concentrated salt– DMSO

– Freezing• expensive

• risky

– Alcohol• Explosive

• Needs regular checking (evaporation)

• degradation

• Store at cold temperature (4oC, -20oC)

– Dried• Extract and purify

• Apply to membranes

• Store dry– Make sure it’s dry

Summary• Tissue collection

– Almost any tissue• Preferably fresh

– Fresh tissue for allozymes

• Tissue storage– Aim: dehydration– Main methods: freezing, alcohol, salt, drying

• DNA extraction– Central theme: cell lysis, protein removal, DNA precipitation– Many variations

• Depending on quality required, throughput and costs

– Commercial kits• Columns or beads

• DNA storage– Freezing, alcohol, salt, drying

Mr. DNA

Laboratory

• Part A – pipetting– Intro to pipettes

– Some exercises

• Part B – Making buffers– Required for DNA extraction

– Work in pairs

• Part C – Tour of MMBL– See facilities

• Part D – set up digestions for Thursday– Two methods

• Salt

• Qiagen DNeasy kit